A large number of protein misfolding diseases are a consequent of the failure of the protein homeostasis or 'proteostasis' network to manage the aberrant fold leading to gain-of-function and loss-of-function diseases (Balch et al. (2008) Science, 319: 916). Cystic Fibrosis (CF) is caused by deletion of Phe 508 in the CFTR protein (deltaF-CFTR). The Phe 508 deletion prevents the CFTR chloride channel from being integrated properly into the proteostasis network resulting in endoplasmic reticulum associated degradation (ERAD) and loss-of-function at the cell surface in multiple tissues including the lung. We have made substantial progress during the previous funding period to develop a proteomic methodologies platform to study CF disease using semi-quantitative multi-dimensional protein identification technology (MudPIT) and absolute quantification strategies using single reaction ion monitoring (SRM). Based on our cumulative results to date we now hypothesize that the Phe508 deletion mutation triggers loss of key protein interactions necessary to stabilize the maturing protein for export and regulation of activity at the cell surface by the proteostasis network. We propose that the proteostasis network can be modified by biologics and chemicals (drugs) to 'repair' deltaF-CFTR function. This competitive renewal will develop and apply quantitative proteomic methods to determine the mechanism of newly discovered chemicals (drugs) and biologics that restore deltaF508-CFTR channel activity at the cell surface. We propose the 3 new Aims that will allow us for the first time to (1) define the biology managing the WT- and deltaF-CFTR protein folds, (2) to characterize the underlying basis for folding mismanagement by the cell in CF disease, and (3) elucidate the adjustments to the proteostasis environment that will allow us to restore (repair) the deltaF-CFTR for function. We will quantitatively evaluate the effect of biologics (Aim 1) and chemicals (drugs) (Aim 2) that modulate CFTR folding, trafficking and function in human lung cells by application of a combination of innovative absolute (SRM) and relative quantitative mass spectrometry (MudPIT) methodologies to quantitatively standardize and define the protein interaction networks (PIN)s required to achieve restoration of deltaF-CFTR channel activity at the cell surface. In Aim 3, using mass spectrometry we will study the basis of deltaF-CFTR function in mouse models of CF disease and a newly developed pig CF model. Mouse models will use of innovative whole animal 15N-labeling technologies. These Aims will provide a proteomic framework to understand the cellular and organismal basis for the clinical restoration of human CF disease. The proposed studies represent the first hypothesis-based application of mass spectrometry to generate a proteomic description of the response of CF disease models to biologics and chemicals that restore cell surface channel activity, and serves as a general model for proteomic analysis of human misfolding disease that is comprehensive in scope. PUBLIC HEALTH RELEVANCE: A large number of protein misfolding diseases are a consequent of the failure of the protein homeostasis or 'proteostasis' network to manage the aberrant fold leading to gain-of-function and loss-of-function diseases. The Phe 508 deletion prevents the CFTR chloride channel (deltaF-CFTR) from being integrated properly into the proteostasis network resulting in loss-of-function at the lung cell surface. This competitive renewal will develop and apply quantitative proteomic methods to determine the mechanism of newly discovered chemicals (drugs) and biologics that restore deltaF-CFTR channel activity at the cell surface through studies in human cell and animal models that will allow us for the first time to (1) define the biology managing the WT- and deltaF-CFTR protein function, (2) to characterize the underlying basis for folding mismanagement by the cell in CF disease, and (3) elucidate the adjustments to the proteostasis environment that will allow us to restore (repair) the deltaF-CFTR for function.